Photokinetic Ocular Drug Delivery Methods and Apparatus

ABSTRACT

The present invention relates generally to transscleral, transcorneal, and transocular delivery of biologically active substances through the tissues, blood vessels and cellular membranes of the eyes of patients without causing damage to the cellular surface, tissue or membrane. The invention provides compositions and methods for enhanced transscleral, transcorneal and transocular delivery of biologically active substances using pulsed incoherent light, and particularly the transcleral, transcorneal or transocular delivery of high molecular weight biologically active substances to a patient using pulsed incoherent light. The invention further provides a device for the application of the pulsed incoherent light to cellular surfaces and membranes of the eye of a subject using those compositions and methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S.application Ser. No. 12/903,126, filed Oct. 12, 2010, published as2012/0125076 on May 26, 2011 and issuing on Feb. 3, 2015 as U.S. Pat.No. 8,948,863, which in turn claims priority to U.S. Provisional PatentApplication Ser. No. 61/250,371, filed Oct. 9, 2009, and U.S.Provisional Patent Application Ser. No. 61/328,625, filed Apr. 27, 2010,all of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The inventions disclosed and taught herein relate generally tophotokinetic delivery of biologically active substances across amammalian ocular surface. More particularly, the invention providesmethods, apparatus, and compositions for the transcleral/transcorneal,ocular delivery of biologically active substances, such as therapeuticagents, using pulsed incoherent light.

BACKGROUND OF THE INVENTION

Millions of people worldwide suffer from ocular diseases, many of whichlead to visual impairment. Anterior segment diseases (dry eye, eye liddiseases) can be successfully treated with topical administration ofdrugs in eye-drop formulation. However, this minimally invasivetechnique only allows for less than about 5%, and many times less than1%, of the administered drug to reach the drug target site before beingwashed away by tear formation or being absorbed systemically by thesurrounding eye tissues. Eye drops may not be an effective method foradministering larger molecular weight drugs into the eye for treatmentof posterior segment eye diseases such as age-related maculardegeneration, diabetic retinopathy, retinitis pigmentosa, and primaryocular lymphoma.

Systemic administration of an eye targeted drug has very poorbioavailability within the eye due to blood-ocular barriers thatnormally protect the eye from circulating antigens, inflammatorymediators, and pathogens. Typically, systemic administration does notyield therapeutic drug levels in the posterior vitreous, retina, orchoroid, and although systemic administration can deliver drugs to theposterior eye, the large systemic doses necessary to achieve intraoculartherapeutic levels are often associated with significant side effects.

As a result of these issues, direct intravitreal drug administration byneedle injection is the current standard of care for many diseases ofthe eye. Recent drug formulation technologies have provided increasedbioavailability and sustained release of drugs that are delivered byintravitreal needle injection. Even with drug formulation advancements,repeated invasive injections are required over extended periods ofmonths and years. Intravitreal administration of drugs by needleinjection is associated with an entirely new set of potentiallycatastrophic side effects such as infection, intravitreal hemorrhage orretinal detachment. Estimates in the literature range from 0.02%(Peyman, et al, Retina, Vol. 29(7), pp. 875-912 (2009)) to 0.2% (Jager,R. D., et al., Invest. Ophthalmol. Vis. Sci., 45 (2004)) which wouldresult in between 200 to 2,000 iatrogenic potentially blinding eyeinfections this year alone. Retinal detachment is estimated at 0.9%prevalence (Jager, 2004) from intravitreal injection, which translatesto approximately 9,000 retinal detachments this year from thisprocedure. Additionally, ocular injections are painful and costly. Manydisease states may require frequent drug administration directly intothe eye to reach and maintain therapeutic levels. An effective,minimally invasive method of intraocular drug delivery is wanting. Theproposed ocular drug delivery system overcomes the problems withrepeated ocular needle injections and may be simple enough for home use.

It is widely recognized that stimulation of the optical sites of organicmolecules result in conformational changes of the molecule which mayproduce a physical change in the shape of the molecule. When thisstimulation is stopped the molecule would then return to a resting stateand original physical shape. Also, if the stimulation is a low power,the relaxational time would be longer than if the molecule wasstimulated with high optical power. The combination of low power andslow cyclical stimulation of the molecular optical sites would result inreversible conformational changes causing the molecule to bend and flexresulting in gross physical movement.

Passive transmembrane permeation is generally time and molecular weight(MW) dependent wherein larger molecules have less permeation flux ratesthan smaller molecules. While not intending to be bound by any theories,it is hypothesized that if a drug molecule in a pharmacologicallyacceptable formulation is placed on the surface of the sclera/cornea andcyclically illuminated with a selected wavelength of light at a selectedpulse rate, the resulting cyclic physical shape change of the moleculemay cause gross movement and result in the migration of the moleculeacross the sclera membrane. It is further hypothesized that narrowwavelength incoherent (non-laser) light from a light emitting diode(LED) source could be used for optical stimulation and that non-ionizingvisible light with these characteristics would not be harmful to thedrug molecule or the sclera itself. Applicants further hypothesize thatthe permeation with this system may be less molecular weight dependentthan with passive transmembrane permeation methodologies.

The penetration of biologically active substances through theintraocular tissues occurs by either passive or active transportmechanisms, typically through the corneal and/or the non-corneal(conjunctival-scleral) pathways. Passive delivery or diffusion relies ona concentration density gradient between the drug at the outer surfaceand the inner surface of the biological barrier to be penetrated. Thediffusion rate is proportional to the gradient and is modulated by amolecule's size, hydrophobicity, hydrophilicity and other physiochemicalproperties as well as the area of the absorptive surface. Typically,topically applied drugs reach the intraocular tissues by either thecorneal and/or the non-corneal (conjunctiva-scleral) pathways, andefforts have been focused on either enhancing transcellular drugpenetration by increasing drug lipophilicity through the use of prodrugsor analogs, or improving paracellular penetration by using enhancers toopen tight junctions (Lee, et al., J. Ocul. Pharmacol, Vol. 2, pp.67-108 (1986)). However, it is common to see about 1% or less of anapplied dose absorbed across the cornea and conjunctiva to reach theanterior segment of the eye (Lee, et al., in RETINA, 3rd Ed., Mosby, St.Louis, pp. 2270-2285 (2001)). Examples of passive delivery systemsinclude ocularly-applied transdermal patches for controlled delivery of,for example, enkephalins, leupeptin (serine protease inhibitor),camostat mesylate (aminopeptidase inhibitor), nitroglycerine (angina),scopolamine (motion sickness), fentanyl (pain control), nicotine(smoking cessation), estrogen (hormone replacement therapy),testosterone (male hypogonadism), clonidine (hypertension), andlidocaine (topical anesthesia). The controlled delivery of these drugscan include the use of polymer matrices, reservoirs containing drugswith rate-controlling membranes and drug-in-adhesive systems.

In contrast, active delivery relies on ionization of the drug or otherpharmacologically active substances and on means for propelling thecharged ions through the tissue. The rate of active transport varieswith the method used to increase movement and propulsion of ions, buttypically this transport provides a faster delivery of biologicallyactive substances than that of passive diffusion. Active transportdelivery systems include methods such as subconjunctival ocular drugdelivery, iontophoresis (transcleral and transcleral/conjunctival), anda variety of other routes which involve carrier-mediated drug transportsystems.

Subconjunctival ocular drug delivery is an active transport method ofattempting to elevate intraocular drug concentrations and minimize thefrequency of dosing. Compared with direct intravitreal injection, thisapproach is less risky to the patient, and less invasive. Since thesclera is much more permeable than conjunctiva, the formidablepermeability barrier consisting of both the cornea and the conjunctivacan be avoided all together with this approach. Advantages ofsubconjunctival ocular drug delivery, such as by the use ofsubconjunctival implants with nano/microparticles and matrix materials,compared to subconjuctival injection of solution, is the achievement ofhigher drug concentrations and sustained release of the drug into boththe vitreous humor and retinal areas (Gilbert, J. A., et al., J.Control. Release, Vol. 89, pp. 409-417 (2003)).

Iontophoresis is a technique used to guide one or more therapeutic ionsin solution into the tissues and blood vessels of the body by means of agalvanic or direct electrical current supplied to wires that areconnected to skin-interfacing electrodes. Although ionotophoresisprovides a method for controlled drug delivery transdermally,irreversible skin damage can occur from galvanic and pH burns resultingfrom electrochemical reactions that occur at the electrode and skininterface. Consequently, its application to ocular therapies has beenlimited, with limited reports of its use in delivering molecules intothe eyes of patients. For example, Asahara reported the use oftransscleral iontophoresis to deliver 6-carboxyfluorescein-labeledphosphorothioate oligonucleotides and a 4.7 kb plasmid that expressedthe green fluorescent protein (GFP) into albino rabbit eyes, with thenucleotides being detected in the anterior chamber, vitreous, andposterior retina with no alteration in length of the oligonucleotides(Asahara, et al., Japn. J. Ophthalmol. Vol. 45, pp. 31-39 (2001)). Morerecently, a low-current, non-invasive iontophoretic treatment usingdexamethasone-loaded hydrogels showed potential value in increasing thedrug penetration to the anterior and posterior segments of the eye. See,Eljarrat-Binstock, E., et al., J. Controlled Release, Vol. 106(3)386-390 (2005); and Myles, M. E., et al., Advanced Drug DeliveryReviews, Vol. 57, pp. 2063-2079 (2005)).

Other approaches to ocular drug delivery problems have included the useof ocular/ophthalmic inserts (e.g., OCUFIT SR®), collagen shields,vesicular systems, the use of liposomes and niosomes, the development ofbioadhesives, mucoadhesive dosage forms, the use of lyophilisate carriersystems, and the use of nanoparticles and microparticles such asnanospheres made up of poly-d,l-lactic acid (PLA),polymethylmethacrylate (PMMA), cellulose, poly-ethyl-caprolactone(PECL), or even chitosan (CS) nanoparticles (DeCampos, et al., Pharm.Res., Vol. 21(5) 803 (2004)) as part of polymeric drug delivery systemsfor drug absorption in the eye. These approaches to drug delivery to theeye have been reviewed extensively in the medical literature. See, Das,S. & Suresh, P. K., Int'l. J. Drug Delivery, 2, pp. 12-21 (2010); and,Sultana, Y., et al., Current Drug Delivery, Vol. 3, pp. 207-217 (2006)).However, many of these approaches suffer limitations as well, such asbeing suitable only for delivering therapeutic molecules of a limitedsize (e.g., molecular weights of less than 200 Da), or unappealing sideaffects or potential for added eye damage for the patient seekingtreatment.

Because of the inherent problems of the above-identified methods, a needexists for a safe and efficient transocular drug delivery method thateliminates side-effects and damage to the barrier function or appearanceof the patient's eye caused by drug administration, and allows for awide range of biologically active substances to be administered by sucha method in therapeutically effective amounts. It would therefore bedesirable to provide compositions, methods, and apparatuses to addressthese problems.

In vitro methods described within the present disclosure were developedto demonstrate the facilitated translocation of two separate compoundsthrough sclera and corneal tissue using pulsed light. These in vitrostudies, as described herein, suggest that the hypotheses proposed bythe applicants been confirmed. The method of ocular drug delivery bypulsed incoherent light as described herein is referred to as“Photokinetic Ocular Drug Delivery” (PODD).

BRIEF SUMMARY OF THE INVENTION

The novel technology described herein generally relates to devices andmethods for transscleral/transcorneal needleless drug administration.Specifically, the technology is an ocular drug delivery method wherein adrug applied to scleral/corneal tissue is illuminated with a selectednarrow wavelength light from a LED source and pulsed at a selectedfrequency that then causes the drug to permeate into and through thetissue. The technology comprises in vitro methods for the selection ofoptical properties of light emitting devices applied to specific drugformulations thus defining in vivo administration systems. Thetechnology provides a non-invasive method using light and drug reservoirdevices to introduce drugs into the eye without the use of needles. Thesystem is safer and less costly than ocular drug administration byneedle injection.

In accordance with one aspect of the present disclosure, a method forthe photokinetic transscleral ocular delivery of a biologically activesubstance to a subject is described, the method comprising preparing asolution comprising the biologically active substance and a solvent;applying the solution to a cellular surface of an eye of the subject;illuminating the solution on the cellular surface with a pulsedincoherent light having a selected wavelength, pulse rate and pulseduration or duty cycle; and allowing the solution to permeate throughthe cellular surface.

In accordance with a further aspect of the present disclosure, a devicefor photokinetic transscleral ocular drug delivery is described, thedevice comprising a generator that provides an oscillating electricalpulse; at least one light emitting diode that receives the oscillatingelectrical pulse and responds by providing an incoherent light; and, adrug reservoir cell that holds a solution comprising a high molecularweight biologically active substance and a solvent; wherein the drugreservoir cell is positioned to receive the incoherent light. In furtheraccordance with this aspect of the disclosure, the generator is anelectrical or repeat cycle square wave pulse generator. In furtheraccordance with this aspect of the disclosure, the device includes alight pad having at least one light emitting diode (LED) embedded withinit.

In accordance with another aspect of the present disclosure, an in-vitromethod of photokinetic transscleral drug delivery to the eye of asubject is described, the method comprising preparing a solutioncomprising a biologically active substance and a solvent; applying thesolution to an ocular cellular surface of a subject; illuminating thesolution on the ocular cellular surface with a pulsed incoherent lighthaving a selected wavelength, pulse rate and pulse duration with adevice; and allowing the solution to permeate through the ocularcellular surface.

In further accordance with aspects of the present disclosure, methodsfor the transcleral delivery of one or more high molecular weightbiologically active substances to the eye of a subject in need of suchtreatment is described, the method comprising preparing a solutioncomprising a high molecular weight biologically active substance and asolvent applying the solution to an ocular cellular surface of asubject; illuminating the solution on the ocular cellular surface with apulsed incoherent light having a selected wavelength, pulse rate andpulse duration with a device; and allowing the solution to permeatethrough the ocular cellular surface. In further accordance with thisaspect of the disclosure, the high molecular weight biologically activesubstance ranges in size from about 100 Da to greater than about 4500kDa. In further aspects of the disclosure, the high molecular weightbiologically active substance has a molecular weight of more than 1,000Daltons, and in still further aspects, the high molecular weightbiologically active substance has a molecular weight of more than 50,000Daltons.

In accordance with yet another aspect of the present disclosure, amethod for the treatment of a subject having a VEGF-related angiogenicdisease affecting the eyes of the subject is described, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a biologically active substance using thetransscleral/transcorneal PODD drug delivery methods described herein,wherein the VEGF-related angiogenic disease is selected from the groupconsisting of cancer, age-related macular degeneration (AMD), anddiabetic retinopathy.

In accordance with a further aspect of the present disclosure, a processfor treating an infection or disorder in a tissue of an eye of a subjectis described, the process of which comprises transclerally ortranscorneally delivering a therapeutic composition to an eye using thephotokinetic ocular drug delivery methods described herein, wherein thetherapeutic composition comprises a biologically active substance havinga molecular weight of at least 500 Daltons, a solvent, and optionally agelling agent.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates an exemplary photokinetic transscleral drug deliverysystem in accordance with the present disclosure.

FIG. 2 illustrates a close-up view of the system of FIG. 1, showing thephotokinetic patch applied to the sclera under the eye lid.

FIG. 3 illustrates a top view of the photokinetic eye patch of FIG. 1.

FIG. 4 illustrates a cross-sectional view through the patch of FIG. 1,taken along line A-A.

FIG. 5 illustrates an exemplary photokinetic eye patch with at least oneLED embedded in the patch material.

FIG. 6 illustrates exemplary Franz diffusion cell apparatus for in vitrodetermination of photokinetic conditions of light wavelength and pulserate.

FIG. 7 illustrates a spectrophotometry light absorbance scan ofmethotrexate (MTX).

FIG. 8 illustrates MTX permeation PODD at 350 nm@24 CPS vs. passivepermeation controls per cm.sup.2 of sclera at 3 time points.

FIG. 9 illustrates MTX permeation PODD at 370 nm@24 CPS vs. passivepermeation controls.

FIG. 10 illustrates PODD Insulin permeation at 405 and 450 nm vs.control for 24 hours at 24 CPS. Herein a 200 IU/mL mixture in a drugcarrier solution was placed in the Franz donor cell. A large differencein permeation is noted here also even within a narrow range of lightwavelength.

FIG. 11 illustrates MTX permeation PODD at 370 nm@24 CPS vs. passivepermeation controls per cm.sup.2 of sclera over a short (60 minute)exposure range.

FIG. 12 illustrates the effect of a variety of wavelengths on permeatingvancomycin through scleral tissue using PODD cells.

FIG. 13 illustrates the effect of 405 and 450 nm wavelength@24 CPSverses passive permeation controls on permeating insulin through scleraltissue using PODD cells.

FIG. 14 illustrates the effect of 405 and 450 nm wavelength@24 CPSversus passive permeation controls on permeating insulin like growthfactor 1 through scleral tissue using PODD cells.

FIG. 15 illustrates the effect of 405 and 450 nm wavelength@24 CPSversus passive permeation controls on permeating Avastin™ throughscleral tissue using PODD cells.

FIG. 16 illustrates the effect of 405 and 450 nm wavelength@24 CPSversus passive permeation controls on permeating hyaluronic acid (HA)through scleral tissue using PODD cells.

FIG. 17 illustrates a summary comparison of the size of biologicallyactive materials which can be transsclerally/transcorneally appliedusing the procedures and apparatus of the present disclosure.

FIG. 18A-18C illustrate rabbit optic disc imaging results of fluorescenttagged human insulin administered with the PODD system of the presentdisclosure.

FIGS. 19 A-F illustrate three concentrations of human FITC labeledinsulin used in the PODD device vs. passive permeation of 4 mU/mL in thevarious fluids and tissues of the rabbit eye (60 minute exposure).

While the inventions disclosed herein are susceptible to variousmodifications and alternative forms, only a few specific embodimentshave been shown by way of example in the drawings and are described indetail below. The figures and detailed descriptions of these specificembodiments are not intended to limit the breadth or scope of theinventive concepts or the appended claims in any manner. Rather, thefigures and detailed written descriptions are provided to illustrate theinventive concepts to a person of ordinary skill in the art and toenable such person to make and use the inventive concepts.

DEFINITIONS

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention. Units, prefixes, and symbols may be denoted in their SIaccepted form. Unless otherwise indicated, nucleic acids are writtenleft to right in 5′ to 3′ orientation, and amino acid sequences arewritten left to right in amino to carboxy orientation, respectively.Numeric ranges are inclusive of the numbers defining the range andinclude each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three letter symbols(e.g., Pro for proline), or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes. Theterms defined below are more fully defined by reference to thespecification as a whole.

The term “subject”, as used herein, refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to humans andother primates, rodents (e.g., mice, rats, and guinea pigs), lagamorphs(e.g., rabbits), bovines (e.g, cattle), ovines (e.g., sheep), caprines(e.g., goats), porcines (e.g., swine), equines (e.g., horses), canines(e.g., dogs), felines (e.g., cats), domestic fowl (e.g., chickens,turkeys, ducks, geese, other gallinaceous birds, etc.), as well as feralor wild animals, including, but not limited to, such animals asungulates (e.g., deer), bear, fish, lagamorphs, rodents, birds, etc. Itis not intended that the term be limited to a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are encompassed by the term.

The term “biologically active substance” refers generally to anychemical, drug, antibiotic, peptide, hormone, protein, DNA, RNA andmixtures thereof that affects biological pathways or interacts withcellular components.

The term “chemical” denotes any naturally found or synthetically madesmall molecule or polymer. A chemical can be a polar (hydrophilic),non-polar (hydrophobic), oleophobic or oleophilic compound. Accordingly,the invention described herein is particularly useful for transport ofcompounds with of high molecular weight, which can be polar, non-polar,oleophobic, including fluorochemicals, and oleophilic, across at leastthe sclera of a patient.

The term “drug” denotes any natural or synthetic compound used fortherapeutic treatment in mammals. Examples of drugs include, but are notlimited to, anti-infective, antibiotic, antifungal, antineoplastics,anti-VEGF, antineovasculars, steroids, anti-inflammatory,immunomodulators, gas, antioxidants, nanoparticles, genes, cytokines,peptides, antithrombotics, nucleotides, RNAs, anti-complimentmedications, compliment modulating medications, peptides,immunoglobulins, antibodies, antigens, anti-glaucoma medications,hormones, vitamins, silicone liquids, heavy liquid tamponades, cellularnutrients, anti-apoptotic agents, anticoagulants, tissue adhesives,cofactors, coenzymes, and enzymes. Specific FDA approved drugs which maybe delivered by the PODD system described herein include, but are notlimited to, triamcinolone acetonide (Kenalog; Bristol Myers Squibb, NewYork, N.Y.), pegaptanib (Macugen; OSI/Eyetech and Pfizer, New York,N.Y.), bevacizumab (Avastin; Genentech, San Francisco); and ranibizumab(Lucentis; Genentech). Numerous other agents available on aninvestigational basis such as VEGF trap (Regeneron; Tarrytown, N.Y.) mayalso be included.

Vitamins are organic chemicals that are essential for nutrition inmammals and are typically classified as fat-soluble or water-soluble.Vitamins required to maintain health in humans include, but are notlimited to, vitamin A (retinol), precursor to vitamin A (carotene),vitamin B.sub.1 (thiamin), vitamin B.sub.2 (riboflavin), vitamin B.sub.3(nicotinic acid), vitamin B (pantothenic acid), vitamin C (ascorbicacid), vitamin D (calciferol), vitamin E (tocopherol), vitamin H(biotin) and vitamin K (napthoquinone derivatives).

The term “antibiotic” refers to any natural or synthetic substance thatinhibits the growth of or destroys microorganisms in the treatment ofinfectious diseases. Although not an exhaustive list, examples ofantibiotics include amoxycillin, ampicillin, penicillin, clavulanicacid, aztreonam, imipenem, streptomycin, gentamicin, vancomycin,clindamycin, cephalothin, erythromycin, polymyxin, bacitracin,amphotericin, nystatin, rifampicin, tetracycline, doxycycline,chloramphenicol and zithromycin.

The term “peptide” refers to a compound that contains 2 to 50 aminoacids and/or imino acids connected to one another. The amino acids canbe selected from the 20 naturally occurring amino acids. The twentyconventional amino acids and their abbreviations follow conventionalusage. See, for example, Immunology—A Synthesis (2.sup.nd Edition, E. S.Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass.(1991)), which is incorporated herein by reference. The amino acids canalso be selected from non-natural amino acids such as those availablefrom Sigma-Aldrich (St. Louis, Mo.), including but not limited toalicyclic amino acids, aromatic amino acids, β-amino acids, γ-aminoacids, norleucine, ornithine, N-methyl amino acids, homo-amino acids,and derivatives of natural amino acids, such as 4-nitro-phenylalanineand xanthenyl-L-asparagine. Although not an exhaustive list, examples ofsuitable peptides include glycine-tyrosine, valine-tyrosine-valine,tyrosine-glycine-glycine-phenylalanine-methionine,tyrosine-glycine-glycine-phenylalanine-leucine and asparticacid-arginine-valine-tyrosine-isoleucine-histidine-proline-phenylalanine.

The term “hormone” refers to a substance that originates in an organ,gland, or part, which is conveyed through the blood to another part ofthe body, stimulating it by chemical action to increased functionalactivity or to increase secretion of another hormone. Although not anexhaustive list, examples of hormones include methionine enkephalinacetate, leucine enkephalin, angiotensin II acetate, β-estradiol, methyltestosterone, progesterone and insulin.

A polypeptide, as used herein, is defined as a chain of greater than 50amino acids and/or imino acids connected to one another.

The term “protein”, as used herein, refers to a large macromoleculecomposed of one or more polypeptide chains. The term “isolated protein”is a protein that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) is free of other proteins from the same species(3) is expressed by a cell from a different species, or (4) does notoccur in nature. Thus, a protein that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The terms DNA and RNA as referred to herein mean deoxyribonucleic acidand ribonucleic acid, respectively. The term “polynucleotide” means apolymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms.

“Gelling agents,” according to the present disclosure, are compoundsthat can behave as reversible or non-reversible networks. Under certainconditions, a gelling agent can be placed in a solvent to form a viscoussolution. Under other conditions, that same gelling agent can be placedin the same or different solvent to form a gel. The role of gellingagents according to the invention is to prevent evaporation loss of thebiologically active substance in the appropriate solvent. Examples ofgelling agents include, but are not limited to, hydroxyethyl cellulose,NATRASOL™, pectines, agar, alginic acid and its salts, guar gum, pectin,polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives,propylene carbonate, polyethylene glycol, hexylene glycol sodiumcarboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropyleneblock copolymers, pluronics, wood wax alcohols and tyloxapol.

The term “solvent” according to the present disclosure is any aqueous ororganic solvent that can be combined with the biologically active agentto form a solution. In one embodiment, the aqueous solvent is water. Inanother embodiment, the solvent can be an aqueous solution of eitherethyl lactate or propylene glycol, both of which act as permeationenhancers. Alternately, the term “solvent” can also mean an adhesiveused to embed a biologically active substance, for example, in a patch.Solvent can also refer to a pharmaceutically-acceptable medium combinedwith the biologically active substance to be used in powder form.

The term “therapeutically effective amount”, as used herein, refers toan amount of an antibody, polypeptide, or other drug effective to“treat” a disease or disorder in a subject or mammal. In the case ofcancer, the therapeutically effective amount of the drug may reduce thenumber of cancer cells; reduce the tumor size; inhibit (i.e., slow tosome extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic.

The phrase “pharmaceutically acceptable salt” as used herein is meant torefer to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well-known in the art. Forexample, P. H. Stahl, et al. describe pharmaceutically acceptable saltsin detail in “Handbook of Pharmaceutical Salts: Properties, Selection,and Use” (Wiley VCH, Zunch, Switzerland: 2002). The salts can beprepared in situ during the final isolation and purification of thecompounds of the present invention or separately by reacting a free basefunction with a suitable organic acid. Representative acid additionsalts include, but are not limited to acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, flimarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups can be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; aryl alkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which canbe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid.

The phrase “pharmaceutical composition” refers to a formulation of acompound and a medium generally accepted in the art for the delivery ofthe biologically active compound to mammals, e.g., humans. Such a mediumincludes all pharmaceutically acceptable carriers, diluents orexcipients therefore.

The phrase “pharmaceutically acceptable carrier, diluent or excipient”as used herein includes without limitation any adjuvant, carrier,excipient, glidant, sweetening agent, diluent, preservative,dye/colorant, flavor enhancer, surfactant, wetting agent, dispersingagent, suspending agent, stabilizer, isotonic agent, solvent, oremulsifier which has been approved by the United States Food and DrugAdministration as being acceptable for use in humans or domesticanimals.

Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest, e.g., tissue injury, in a mammal,preferably a human, having the disease or condition of interest, andincludes: (i) preventing the disease or condition from occurring in amammal, in particular, when such mammal is predisposed to the conditionbut has not yet been diagnosed as having it; (ii) inhibiting the diseaseor condition, i.e., arresting its development; (iii) relieving thedisease or condition, i.e., causing regression of the disease orcondition; or (iv) relieving the symptoms resulting from the disease orcondition.

As used herein, the terms “disease,” “disorder,” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

As used herein, the term “%” when used without qualification (as withw/v, v/v, or w/w) means % weight-in-volume for solutions of solids inliquids (w/v), % weight-in-volume for solutions of gases in liquids(w/v), % volume-in-volume for solutions of liquids in liquids (v/v) andweight-in-weight for mixtures of solids and semisolids (w/w), such asdescribed in Remington's Pharmaceutical Sciences [Troy, David B., Ed.;Lippincott, Williams and Wilkins; 21st Edition, (2005)].

The term “drug” as used in conjunction with the present disclosure meansany compound which is biologically active, e.g., exhibits or is capableof exhibiting a therapeutic or prophylactic effect in vivo, or abiological effect in vitro.

The term “donor solution” or “delivery medium” comprises thebiologically active substance itself or any mixture of this substancewith a solvent, a gelling agent, a photocatalytic agent, a carrier oradjuvant, a skin-penetrating agent, a membrane-penetrating agent andcombinations thereof. The biologically active substance, or alternately“active ingredient” does not have to be dissolved in a solvent but canbe suspended or emulsified in a solvent. The donor solution or deliverymedium can take the form of an aqueous or an organic liquid, a cream, apaste, a powder or a patch.

Although not an exhaustive list, examples illustrating the term “mammal”include human, ape, monkey, rat, pig, dog, rabbit, cat, cow, horse,mouse, and goat Skin surfaces or membranes according to the inventionrefer to those of a human or other mammal.

The term “viscous solution” refers to a solution that has an increasedresistance to flow.

The term “cellular surface” refers to an outer layer of the skin, a cellmembrane, or tissue.

The term “transmembrane” refers to the penetration and movement of abiologically active substance from an extracellular environment to anintracellular environment.

The term “transocular”, as used herein, refers to the penetration andmovement of a biologically active substance from an external region ofthe eye of a subject to the interior region of the eye of the subject.

The term “incoherent light” refers to electromagnetic waves that areunorganized and propagate with different phases. The term “pulsedincoherent light” is any incoherent light having a discrete ON and OFFperiod.

In contrast, “coherent light” refers to all light rays that are in phaseand oriented in the exact same direction to produce a concentrated beamof light. Lasers generate these types of rays and can penetrate throughmaterials such as solid media, including metals (e.g., sheet metal).

The term “light emitting diode” or “(LED)” as used herein refers to adevice that generally emits incoherent light when an electric voltage isapplied across it. Most LEDs emit monochromatic light at a singlewavelength that is out of phase with each other. According to theinvention, most, if not all, types of LEDs can be used. For example, anLED having output range from red (approximately 700 nm) to blue-violet(approximately 350 nm) can be used. Similarly, infrared-emitting diodes(IRED) which emit infrared (IR) energy at 830 nm or longer can also beused.

The terms “optically clear medium” or “light pad” as used herein referto materials that act as a filter to all wavelengths except thosewavelengths emitted from a light source. In a preferred embodiment ofthe present disclosure, the light pad is comprised of clear poly(methylmethacrylate) or clear silicon rubber.

The term “reflective coating or layer” as used herein is a material thatis coated on at least one surface of the light pad. Those skilled in theart will appreciate that the reflective layer can be a wavelengthspecific reflective coating (e.g., aluminum, ZnO, silver or anyreflective paint).

The term “photokinetic” as used herein refers to a change in the rate ofmotion in response to light, as an increase or decrease in motility witha change in illumination.

DETAILED DESCRIPTION

The Figures described above and the written description of specificstructures and functions below are not presented to limit the scope ofwhat Applicants have invented or the scope of the appended claims.Rather, the Figures and written description are provided to teach anyperson skilled in the art to make and use the inventions for whichpatent protection is sought. Those skilled in the art will appreciatethat not all features of a commercial embodiment of the inventions aredescribed or shown for the sake of clarity and understanding. Persons ofskill in this art will also appreciate that the development of an actualcommercial embodiment incorporating aspects of the present inventionswill require numerous implementation-specific decisions to achieve thedeveloper's ultimate goal for the commercial embodiment. Suchimplementation-specific decisions may include, and likely are notlimited to, compliance with system-related, business-related,government-related and other constraints, which may vary by specificimplementation, location and from time to time. While a developer'sefforts might be complex and time-consuming in an absolute sense, suchefforts would be, nevertheless, a routine undertaking for those of skillin this art having benefit of this disclosure. It must be understoodthat the inventions disclosed and taught herein are susceptible tonumerous and various modifications and alternative forms. Lastly, theuse of a singular term, such as, but not limited to, “a,” is notintended as limiting of the number of items. Also, the use of relationalterms, such as, but not limited to, “top,” “bottom,” “left,” “right,”“upper,” “lower,” “down,” “up,” “side,” and the like are used in thewritten description for clarity in specific reference to the Figures andare not intended to limit the scope of the invention or the appendedclaims.

Computer programs for use with or by the embodiments disclosed hereinmay be written in an object oriented programming language, conventionalprocedural programming language, or lower-level code, such as assemblylanguage and/or microcode. The program may be executed entirely on asingle processor and/or across multiple processors, as a stand-alonesoftware package or as part of another software package.

Applicants have created methods, apparatus, and systems for thetranscleral/transcorneal ocular delivery of biologically activemolecules and compositions to a mammal, using photokinetic deliverymethods and assemblies.

One embodiment of the invention relates to compositions for photokinetictranscleral/transcorneal delivery, also referred to herein asPhotokinetic Ocular Drug Delivery (PODD) of one or more biologicallyactive substances to and through the tissues of a patient's eyes, usingpreferably pulsed incoherent light or, alternatively, regulated coherentlight. The composition may comprise a biologically active substance asthe delivery medium.

The composition may alternatively comprise a biologically activesubstance and a solvent. The percent of biologically active substance insolvent can be in the range of between 0.0001 to 99.9999% (w/v).Preferably, the biologically active substance is present in aconcentration range of between about 0.01% to about 2% (w/v). Morepreferably, the biologically active substance is present in aconcentration range of between about 0.1 mg/ml to about 10 mg/ml in thesolvent or, alternatively, between about 0.01% to about 1% (w/v). Due tothe high level of permeation achieved by the methods and devicesdescribed herein, low concentrations of a biologically active substancein solvent or in other compositions described herein can be used forefficient transcleral or transcorneal delivery.

The composition may instead comprise a biologically active substance, agelling agent and a solvent. The percent gelling agent in a solution ofbiologically active substance can vary depending on the type of gellingagent used. For example, Klucel is typically used at 1% (w/v), Natrasolat 1.5% (w/v), Carbopol at 0.75% (w/v), and TEA at 0.25% (w/v).

The biologically active substance of the above compositions for use intranscleral or transcorneal administration to a subject for therapeuticpurposes may be selected from the group consisting of chemicals, drugs,antibiotics, peptides, hormones, proteins, DNA, RNA and mixturesthereof. Preferably, in accordance with one aspect of the presentdisclosure, the biologically active substances which may be used fortranscleral/transcorneal, non-invasive delivery to the eye of a subjectare large molecules. As used herein, the phrase “large molecules” asapplied to biologically active substances refers to those biologicalsubstances having molecular weights of at least 100 Daltons (Da),preferably more than about 500 Daltons (Da), more preferably more thanabout 1,000 Daltons (Da), and even more preferably a molecular weight ofmore than about 5,000 Daltons (Da), such as molecular weights of about50,000 Daltons or greater, including compounds having molecular weightsof about 100,000 Daltons (Da) or more, such as compounds havingmolecular weights of about 150,000 Daltons (Da), e.g., about 149 kDa inthe case of infliximab (REMICADE©). For example, the biologically activesubstances which may be therapeutically administered to a subject usingthe PODD methods of the present disclosure may be large molecules havinga molecular weight ranging from about 100 Da to about 150,000 Da, orranging from about 500 Da to about 150,000 Da, as well as ranges withinthis range, such as from about 1,000 Da to about 130,000 Da, from about5,000 Da to about 125,000 Da, and from about 10,000 Da to about 100,000Da, as well as ranges within these ranges, such as from about 20,000 Dato about 135 Da, inclusive. In accordance with the present disclosure,as used herein, the molecular weight of a molecule refers to the sum ofthe weights of the atoms of which it is made, typically abbreviated as“MW” or “mw”, and herein typically expressed in Daltons (Da).

The drug may be selected from the group consisting of anti-infectives,antibiotics, antifungals, antivirals, antineoplastics, anti-VEGFs,antineovasculars, steroids, anti-inflammatories (including NSAIDS),immunomodulators, gases, antioxidants, nanoparticles, genes, cytokines,peptides, antithrombotics, nucleotides, RNAs, anti-complimentmedications, compliment modulating medications, peptides,immunoglobulins, antibodies, antigens, anti-glaucoma medications,hormones, vitamins (such as cyanocobalamin, vitamin B.sub.12), aminoacids, silicone liquids, heavy liquid tamponades, cellular nutrients,anti-apoptotic agents, anticoagulants, tissue adhesives, cofactors,coenzymes, and enzymes. In a preferred embodiment of the presentdisclosure, the drug is an anesthetic, preferably lidocaine.

The compositions according to the invention may also compriseantibiotics as the biologically active substance. Antibiotics accordingto the invention are selected from the group consisting of amoxycillin,ampicillin, penicillin, clavulanic acid, aztreonam, imipenem,streptomycin, gentamicin, vancomycin, clindamycin, cephalothin,erythromycin, polymyxin, bacitracin, amphotericin, nystatin, rifampicin,tetracycline, doxycycline, chloramphenicol, tobramycin, and zithromycin.In a preferred aspect of this embodiment, the biologically activesubstance is the antibiotic vancomycin.

The compositions according to the invention may also comprise antiviralsas the biologically active substance. Antivirals according to theinvention are selected from the group consisting of guanosinederivatives, nucleoside phosphonates, phosphonic acid derivatives,oligonucleotides, and combinations thereof, as well as pharmaceuticallyacceptable salts, solvates, hydrates, and derivatives thereof. In apreferred aspect of this embodiment of the present disclosure, theantiviral is foscarnet (FOSCAVIR™, Astra Zeneca), fomivirsen sodium(VITRAVENE™, Isis Pharmaceuticals), trifluridine (VIROPTIC™),ganciclovir, cidofovir, and vidarabine (Vira-A™, MonarchPharmaceuticals).

Similarly, in another embodiment of the present disclosure, thebiologically active substance is a peptide selected from the groupconsisting of known biologically active peptides, including but notlimited to antibiotic peptides, antifungal peptides, anticancerpeptides, immunological and inflammatory peptides, opioid peptides,neurotrophic peptides, and the like. In a preferred embodiment of thepresent disclosure, the peptide is insulin-like growth factor-1 (IGF-1;also known as somatomedin C or mechano growth factor and having amolecular weight of 7649 daltons).

In further embodiments of the present disclosure, the biologicallyactive substance to be transsclerally delivered using the PODD systemdescribed herein is a hormone or steroid, or a pharmaceuticallyacceptable salt, solvate, hydrate, or derivative thereof. The hormoneswhich may be used for therapeutic applications in accordance with thisdisclosure are selected from the group consisting of methionineenkephalin acetate, leucine enkephalin, angiotensin II acetate,β-estradiol, methyl testosterone, methyl prednisolone, corticosteroids(including corticosone, cortisone, hydrocortisone, and aldosterone),budenoside, progesterone, and insulin. In accordance with one particularaspect of this embodiment, the biologically active hormone is insulin.In accordance with another aspect of this embodiment, the biologicallyactive substance is the steroid triamcinolone acetonide (TA; KENALOG™,Bristol Myers Squibb).

In another embodiment of the present disclosure, the biologically activesubstance to be transsclerally or transcorneally delivered to a subjectusing the PODD system described herein is a protein. The protein may beselected from the group consisting of enzymes, non-enzymes, antibodies(including monoclonal antibodies), and glycoproteins. In one embodimentof the invention, the protein is a humanized antibody. In accordancewith yet another aspect of this embodiment of the present disclosure,the biologically active substance to be transclerally or transcorneallydelivered to a subject is a fusion protein, such as VEGF Trap-Eye (aninvestigational drug manufactured by Regeneron, Tarrytown, N.Y.). Inaccordance with a further aspect of this embodiment of the presentdisclosure, the protein is a monoclonal antibody selected from the groupconsisting of adalimumab (Humira™; Abbott), bevacizumab (Avastin™;Genentech), daclizumab (Zenapax™; Roche); etanercept (Enbril™; Amgen);infliximab (Remicade™; Centocor); ranibizumab (LUCENTIS®; Genentech);and rituximab (Rituxican™; Genentech).

In another embodiment of the present disclosure, the biologically activesubstance to be transclerally or transcorneally delivered to a subjectusing the PODD system described herein is an anticancer agent such as5-fluorouracil (5-FU), mitomycin C, melphalan, carboplatin,methotrexate, or colchicine, or a compound for the treatment ofneovascular (wet) age-related macular degeneration (AMD), such aspegaptanib (MACUGEN™, OSI/Eyetech and Pfizer).

Compositions according to the present disclosure can also contain agelling agent in combination with the biologically active agent and/orsolvent. The gelling agent may be selected from the group consisting ofhydroxyethyl cellulose, Natrasol™, pectines, agar, alginic acid and itssalts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide,cellulose and its derivatives, propylene carbonate, polyethylene glycol,hexylene glycol sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene block copolymers, pluronics, wood waxalcohols, and tyloxapol. In a preferred embodiment of the presentdisclosure, the gelling agent is hydroxypropyl cellulose.

The compositions for therapeutic transscleral/transcorneal deliveryusing the methods and devices described herein may also comprise asolvent that is an aqueous solvent, an organic solvent, or a mixturethereof (such as oil-in-water micro-emulsions) as appropriate. Inaccordance with one embodiment, the aqueous solvent is water. In yetanother embodiment, the aqueous solvent is an aqueous solution of ethyllactate or propylene glycol. Preferably, the water is HPLC grade orpurified by means such as reverse osmosis or distillation. In accordancewith further embodiments of the disclosure, the solvent is an organicsolvent selected from the group consisting of dimethylsulfoxide (DMSO)and poly(ethylene oxide)s (PEOs).

The donor solution or delivery medium according to the invention iscomprised of a biologically active substance itself or any mixture of abiologically active substance with a solvent, a gelling agent, a carrieror adjuvant, a tissue-penetrating agent, emulsifier, one or moredifferent biologically active substances, polymers, excipients, coatingsand combinations thereof. In essence, the biologically active substanceor substances can be combined with any combination of pharmaceuticallyacceptable components to be delivered to the cellular surface by themethod described herein, e.g., photokinetic transscleral and/ortranscorneal ocular delivery. The biologically active substance does nothave to be dissolved in a solvent but can be suspended or emulsified ina solvent. The donor solution or delivery medium can take the form of anaqueous or an organic liquid, a cream, a paste, a powder, or a patch.The donor solution can also comprise microspheres or nanospheres ofbiologically active substances.

Turning now to the Figures, FIG. 1 illustrates an intended applicationfor a photokinetic transscleral and/or transcorneal drug deliveryapplication in accordance with the present disclosure. FIG. 2illustrates a closer view of the photokinetic patch of FIG. 1, showingthe attachment of the patch to the sclera under the eyelid. FIG. 3illustrates a top view of an exemplary photokinetic eye patch inaccordance with the present disclosure. FIG. 4 illustrates across-section through the patch of FIG. 3. FIG. 5 illustrates anexemplary photokinetic eye patch in accordance with the presentdisclosure in combination with an LED embedded in the patch material.These figures will be discussed in combination.

In FIG. 1, a general illustration of an intended application forphotokinetic transscleral and/or transcorneal drug delivery inaccordance with aspects of the present disclosure is shown, wherein aphotokinetic transcleral drug delivery system is illustrated, comprisinga patch, a square pulse generator, and a transmission wire connectingthe two. The photokinetic patch 1 is applied to the exterior surface ofthe eye of a subject, in this case a human. An electronic pulse isgenerated by any suitable square wave pulse generator 20, which may bemounted over the ear or in other suitable manners (e.g., in a headband)proximate to the eye of the subject to be treated. This electrical pulsesignal is transmitted to the photokinetic patch via an electric currenttransmitting wire 5. FIG. 2 illustrates an enlarged region of thesubject's eye of the system of FIG. 1, showing the photokinetic patch 1applied to the sclera 7 of the subject's eye, under the eyelid 8. Thepatch 1 includes a patch material, at least one LED associated with thepatch, and electrical conducting wires 4. The patch material 2 may beany optically clear material that is flexible and soft enough to beapplied to the eye without causing physical damage to the tissue. Thepatch is positioned so that the light output is directed in thedirection of the eye tissue.

FIG. 3 illustrates a top view of the photokinetic eye patch 1 shown inFIGS. 1 and 2. As illustrated therein, the optically clear patchmaterial 2 includes at least one LED 3 embedded within the patchmaterial 2, although it is envisioned that the patch may include aplurality of LEDs, as appropriate. In the event that there are aplurality (two or more) LEDs 3 embedded in the material, the LEDs may beinterconnected within the patch itself, and then connected to a leadwire 5 coming from the electrical square wave pulse generator 20. FIG. 4illustrates a cross-sectional view of the patch 1 of FIG. 3 taken alongline A-A. As is evident from this view, the patch is preferablymanufactured with at least a slight curvature, so as to accommodate thenormal curvature of the eye. Such curvature may be a predefined,standard curvature, or may be individually crafted for individualpatients, depending upon their needs. As is also evident from FIG. 4,the LEDs 3 are embedded within the patch, and are electrically connectedvia communication wire 4, and are in turn connected to a conductor leadwire 5 coming from the wave pulse generator. The side of the patch 9that contacts the eye, and which is opposite the exterior face 11 of thepatch, in accordance with aspects of the present disclosure, is coatedwith a drug layer 6 in such a manner that the drug within the drug layer6 comes into contact with the surface of the subject's eye. This druglayer 6 is preferably positioned between the eye tissue and the LED 3,so that the drug layer 6 receives the light generated by the LED duringoperation of the apparatus, so as to allow the drug within the layer 6to transsclerally permeate the eye for therapeutic purposes.

FIG. 5 illustrates an alternative arrangement of the system of FIG. 1,wherein the photokinetic eye patch 1 includes at least one LED 3embedded within the patch material 2, with electrical conducting wires 4electrically attached to a wire in communication with the pulsegenerator 5.

FIG. 6 illustrates an exemplary Franz diffusion cell apparatus for invitro determination of photokinetic conditions of light wavelength andpulse rate, in accordance with the present disclosure. Franz diffusioncells 31 are shown within a heat block 25, one (B) being shown inpartial cut-away for purposes of clarity.

The testing device illustrated in FIG. 6, in accordance with the presentdisclosure, provides photokinetic transscleral and transocular deliveryof biologically active substances to a portion of an eye by illuminatinga biologically active substance with pulsed incoherent light. Testingdevice can include a light source (not shown) that illuminates abiologically active substance in donor chamber 21 such that thebiologically active substance diffuses into the eye tissue 23 withlittle to no damage to the eye tissue 23. Testing device can also bearranged such that the light source illuminating a biologically activesubstance in donor chamber 21 is horizontal or parallel to a surface onwhich it is mounted.

Testing device may include an electrical driver circuit that providescontrol signals to the light source such that pulsed incoherent light isprovided to the donor chamber 21. The driver circuit may also providecontrol signals that control the intensity, direction, and/or frequencyof the light source. A pulsed incoherent light advantageously andcyclically illuminates the subject drug formulation 24 producing aperiod of excitation and relaxation of the drug 24 and the eye tissue 23which provides photokinetic transscleral and transocular translocationof biologically active substances within donor cell 21 into and throughthe eye tissue 23.

Electronic Driver circuit may regulate an electrical signal that turns(i.e., switches) light source ON and OFF at a particular frequency. Suchan electrical signal may be provided, for example, by a voltagegenerator controlled by an electronic flasher circuit. Alternatively, adriver circuit may itself be a voltage generator and may produce anelectrical signal to control the switching characteristics of lightsource. For example, a voltage generator coupled to light source mayprovide an electrical square wave to power the light source. This squarewave may have a desired ON and OFF period such that light sourceprovides pulsed incoherent light with a desired pulse frequency (e.g., asquare wave period of 0.5 seconds ON and a 0.5 seconds OFF would causelight source to switch at 1 Hz or 1 cycle per second (CPS)).

The light source preferably provides incoherent light (to reduce thepossible damage done to eye tissue 23 or cause damage to the drug 24during the use of testing device). The light source may be, for example,an LED, halogen light source, fluorescent light source, natural light,or other source of light. More particularly, the light source can be alight emitting diode (LED) (fluorescence, 350-1700 nm) or an infraredlight emitting diode (ILED) or a Mercury-Argon (253-922 nm), pulsedxenon (UV-VIS, 200-1000 nm), deuterium (UV, 200-400 nm),deuterium/halogen (UV/VIS/NIR, 200-1700 nm) or tungsten halogen(color/VIS/NIR, 360-1700 nm) light source. The light source preferablyis operable in the range from red (approximately 700 nm) to blue-violet(approximately 350 nm). Similarly, infrared-emitting diodes (IREDs) thatemit infrared energy at 830 nm or longer may be used.

The light source does not have to be an incoherent light source. Inaccordance with aspects of the present disclosure, the light source maybe a coherent light source such as, for example, a laser. In that case,the driver circuit, or other regulation circuitry, is preferably used toturn the coherent light source ON and OFF to reduce the amount of damageto eye tissue 23 while still photokinetically delivering a biologicallyactive substance 24 from the donor cell 21 into the eye tissue 23.Furthermore, a light regulation/conversion device may be placed betweena coherent light source in the donor cell 21 to convert the coherentlight to incoherent light.

Note that a device such as an electronic driver circuit or a controlledvoltage generator is not required to pulse light source. Alternatively,a mechanical shutter may be employed between light source and donorcell. Such a shutter selectively OPENs and CLOSEs such that donor cellis supplied pulsed incoherent light from light source. The speed atwhich the shutter OPENs and CLOSEs determines the frequency of the lightpulsed onto the eye tissue. Filters (not shown) may also be placedbetween light source and the donor cell in order to remove, for example,light of specific wavelengths that may damage the eye tissue or reducephotokinetic activity. Alternatively, the light source may not need beimmersed or optically coupled with the drug solution found in donorcell. The essential arrangement is when a drug in contact with thesubject's eye tissue is positioned to receive pulsed incoherent lightfrom a selected source at a selected pulse frequency.

Preferably, the wavelength of light reaching eye tissue is chosen notonly to reduce damage to the tissue, but also to increase thephotokinetic activity in donor cell (e.g., 350 nm to 450 nm). The pulserate of such light may also be between 1.7 cycles per second (cps) and120 cps (e.g., 24 cps). If fluorescent light is employed as lightsource, it preferably has a wavelength range from about 260 nm to about760 nm. If ultraviolet, visible, near infrared, or halogen light isemployed as light source, the light source preferably has a wavelengthrange from about 340 nm to about 900 nm. The invention is not limited tothese wavelengths. Any method to pulse illuminate the drug that is incontact with the eye tissue may provide the photokinetic transoculardrug delivery.

Donor chamber 21 holds a biologically active substance (e.g., chemicals,drugs, antibiotics, peptides, hormones, proteins, DNA, RNA and mixturesthereof). Donor chamber 21 may also include a solvent that forms asolution with the biologically active substance. The solution may alsoinclude a gelling agent, as appropriate. The solvent may be an aqueousor an organic solvent. Furthermore, eye tissue 23 may be a cellularsurface which is any layer of an eye, such as sclera, cornea or othereye tissue of a mammal. Generally, eye tissue 23 may be any medium thatallows at least the biologically active portion drug formulation 24contained in the donor chamber 21 to diffuse into that medium inresponse to that medium being exposed to a selected light source pulsedat a selected pulse rate. In one embodiment, this medium is a sclera fortransscleral delivery. In another embodiment the medium is cornealtissue for transcorneal delivery.

A clamp (not shown) may optionally be included in testing device tocouple donor chamber 21 and eye tissue 23 to the recipient chamber 22.Drug components comprising the donor formulation 24 placed in donorchamber 21 may be present in recipient chamber 22 as a result of thediffusion of at least the biologically active portion 24 of donorchamber 21 through eye tissue 23. Also, recipient chamber 22 may containa solvent, e.g., HPLC grade water, wherein diffusion of at least thebiologically active portion 24 of donor cell 21 through eye tissue 23enters into the solvent. Generally, the concentration of thebiologically active substance is higher in donor chamber 21 than inrecipient chamber 22.

Temperature control device, such as a heat block, is preferably appliedto at least a portion of the recipient chamber 22. Temperature directorsmay be included as a part of heat block 25 or coupled to the recipientchamber 22 to direct temperature control device 25. Temperaturedirectors (not shown) may also be used to structurally provide supportfor a heat source such as a heat bath. For example, warm water may beplaced in housing defined by temperature directors and a portion ofrecipient chamber 22 between temperature directors. Further to thisexample, a heat source may be used to heat such water. Alternatively, aheat source may be directly coupled to recipient chamber 22. Preferably,temperature control device 25 heats the Franz cell assembly 31 to aconstant level. While the temperature of the solvent in recipientchamber 22 can vary, it is preferably about 37° C., human bodytemperature, or about 35.5° C., human eye surface temperature. Forapplications requiring Franz cell assembly 31 to be cooled, temperaturecontrol device 25 may additionally or alternatively be a cooling source.A temperature sensor (not shown) may be placed in, on, or about theFranz cell 31 or a heat source such that temperature control device 25keeps the Franz cell 31 at a particular temperature for a particularperiod of time.

With continued reference to FIG. 6, stir bar 26 may be included inrecipient chamber 22 to stir any solution in recipient chamber 22.Preferably, stir bar 26 constantly stirs the solution in recipientchamber 22. Recipient chamber 22 may be alternatively stirred, forexample, by a shaking device. Removal of stir bar 26 would, for example,recipient chamber 22 to be easily sanitized while reducing the designcomplexity of recipient chamber 22 assembly. Stir bar 26 may beconnected to an electrical motor (not shown).

Side arm port 27 may be included in recipient chamber 22 to add orremove samples to or from recipient chamber 22 or solutions to or fromrecipient chamber 22. Generally, port 27 is an aperture into recipientchamber 22. An alternate guide tube (not shown) may be included to forman extended port 27 such that a sample recovery or dispersal tool caneasily migrate to port 27. A cover may be employed on port 27 such thatcontaminants from outside recipient chamber 22 do not pass through port27 when samples are being added or removed from recipient chamber 22. Inaccordance with certain aspects of the present disclosure, if a guidetube is included in association with port 27, the guide tube isgenerally an adapter. For example, if the recovery/dispersal tool is aneedle, then guide tube 27 preferably facilitates the coupling of theneedle to port 27.

The Franz cell apparatuses 31 are designated to determine passivepermeation A or photokinetic permeation B into and through eye tissues23. The Franz cell has two chambers—the donor chamber 21 and therecipient chamber 22. Eye tissue 23 is placed between the two chambersand sealed into place and held between the two chambers by a clamp (notshown). The recipient chamber 22 is filled with an aqueous solutionselected to allow for chemical analytical methods. The donor chamber 21is filled with a drug in a pharmacologically acceptable formulation, asdescribed herein. The recipient chamber 22 is constantly stirred by amagnetic stir bar 26. A portion of the recipient chamber is placed in aheat block 25 heated to a physiological temperature (about 35.5° C.). Atvarious time points, samples are dawn from the side arm port 27 forpurposes of chemical analysis.

The passive permeation cell A provides permeation flux rates though thescleral tissue. In the photokinetic Franz cell B, a selected LED 28 ispartially submerged within the drug formulation 24 within the donorchamber 21. The LED is driven by an external pulse generator at aselected pulse rate and connected to the LED electrical connectors 29.The scleral tissue 23 is positioned in contact with and under the drugformulation 24. The drug formulation in contact with the tissue isilluminated by the light 30 generated by the LED 28.

The photokinetic transocular drug delivery methods described herein areuseful for the therapeutic treatment of a variety of ocular disorders bydelivering a wide variety of drugs of a large variety of molecularweight ranges into and through the scleral tissue of the patient. Oculardiseases and disorders suitable for therapeutic treatment with themethods and systems described herein include but are not limited tocancer, such as primary ocular lymphoma; diabetic retinopathy, includingproliferative diabetic retinopathy (PDR); diabetic retinoblastoma;diabetic macular edema; macular degeneration, including “wet”(exudative) macular degeneration and age-related macular degeneration(AMD); intraocular edematous; uveitis, including posterior uveitis;inflammatory diseases; retinitis; glaucoma, including neovascularglaucoma; cicatrizing conjunctivitis; myasthenia gravis; macular edema;choroidal neovascularization; endophthalmitis; ocular toxoplasmosis; andproliferative vitreous retinopathy (PVR). In accordance with aspects ofthe present disclosure, the transocular drug delivery methods describedherein are useful in the treatment of diabetic retinopathy, macularedema, and diabetic retinoblastoma. In accordance with further aspectsof the present disclosure, the transocular photokinetic ocular drugdelivery (PODD) methods and systems described herein are useful in thetreatment of glaucoma, including neovascular glaucoma. In accordancewith still further aspects of the present disclosure, the transocularphotokinetic ocular drug delivery (PODD) methods and systems describedherein are useful in the treatment of uveitis and ocular inflammatorydiseases. In accordance with another aspect of the present disclosure,the transocular photokinetic ocular drug delivery (PODD) methods andsystems described herein are useful in the treatment of maculardegeneration, including both wet macular degeneration and age-relatedmacular degeneration.

In accordance with the treatment of ocular disorders and diseases usingthe systems and methods described herein, the methods of treatment forany of the diseases and disorders set forth above, particularlyglaucoma, diabetic retinopathy, uveitis, and macular degeneration,comprise administering a therapeutically effective amount of a compound,preferably a high-molecular weight compound, or a pharmaceuticallyacceptable salt, solvate, hydrate, racemate, or stereoisomer thereof, toa subject in need thereof using the PODD methods described herein. Forexample, the instant disclosure envisions methods for the treatment ofglaucoma comprising administering a therapeutically effective amount ofa compound, preferably a high-molecular weight compound, or apharmaceutically acceptable salt, solvate, hydrate, racemate, orstereoisomer thereof, to a subject in need thereof. Similarly, theinstant disclosure envisions methods for the treatment of uveitis,diabetic retinopathy, or macular degeneration in a patient wherein themethod comprises administering a therapeutically effective amount of acompound, preferably a high-molecular weight compound, or apharmaceutically acceptable salt, solvate, hydrate, racemate, orstereoisomer thereof, to a subject in need thereof using the PODDmethods described herein. Further, the instant disclosure envisionsmethods for the treatment of VEGF-related angiogenic diseases,particularly those selected from the group consisting of cancer,age-related macular degeneration (AMD), and diabetic retinopathy,wherein the method comprises administering a therapeutically effectiveamount of a compound, preferably a high-molecular weight compound, or apharmaceutically acceptable salt, solvate, hydrate, racemate, orstereoisomer thereof, to a subject in need thereof using the PODDmethods described herein.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor(s) to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 Methotrexate Transcleral Delivery

Materials and Methods

A traditional “up and down” vertical Franz cell skin perfusion apparatusas described above was adapted and used for this sclera tissuepermeation model. The absorption spectrum of the test drug, methotrexate(MTX, a drug used in the treatment of primary ocular lymphoma) wasdetermined. Two wavelengths of light were selected of this facilitatedpermeation study. Samples of the Franz recipient solution were taken at15, 30 and 60 minutes of photokinetic exposure and passive controls andanalyzed by HPLC.

Instruments and Materials

Franz cells (PermeGear, Inc, Bethlehem Pa.) having an 11.28 mm diameterpermeation area, 1.0 cm² were used in the MTX experiment. A photokineticocular drug delivery (PODD)-modified Franz cell testing device wasconfigured so that it accommodated the placement of LEDs within thedonor chamber. The cells were placed within an aluminum block heater35.5° C. on a magnetic stir bar setup (Custom manufactured by PermeGear,Inc, Bethlehem Pa.).

Spectrophotometry measurements (Beckman DU 650) of Methotrexate (SigmaChemicals) (FIG. 7) showed a minor peak of absorption at 370 nm in thenear visible light range.

Discrete wavelength LEDs were purchased from Roithner LasertechnickGmbH, Vienna, Austria as follows: 351 nm, #RTL350-30; 370 nm,#RLS-UV370. Peak emitting wavelengths are ±10 nm at the 50% radianceoutput. The LEDs were driven by a square wave pulse generator built bythe investigators for this study. The adjustable square wave pulsegenerator provided pulsed electrical energy set at 24 cycles per second(CPS) with a 50% pulse duration (ON 50%, OFF 50% of the time). Drivercurrent to the LEDs was limited to or slightly below the manufacturersspecified drive current level to avoid exogenous heat generation. Theexperimental arrangement is shown generally in FIG. 6. As shown therein,Franz cells 31 are adapted for sclera photokinetic permeation studies. Adonor cell contains the test drug (in this example, methotrexate (MTX))in a solvent formulation. The recipient cell is filled with a balancesalt solution. Samples for chemical analysis are taken from the side arm27 of the Franz apparatus as discussed above. Control cells are set upthe same, but without the LED. LEDs are driven by a square wave pulsegenerator (not shown) at a voltage slightly less than the manufacture'sspecified voltage.

Sclera: Ovine sclera was used as the subject tissue, and was selectedbecause ovine sclera is slightly thicker than human or pig sclera, andso the total drug flux may be less than what could be attained in humanuse. Ovine eyes were procured by enucleation within one hour aftereuthanasia and placed in tissue culture medium with antibiotics andantimycotics and refrigerated at 4° C. Sclera tissue was dissected fromthe eyes and placed between the donor and recipient chambers of theFranz apparatus. Sclera was stored for a maximum of 30 hours in thetissue culture at 4° C. before the start of the experiment.

Drug Formulation: Methotrexate (MTX; (4-Amino-10-methylfolic acidhydrate); available from Sigma-Aldrich Corp., St. Louis, Mo.) wasdissolved in a permeation enhancement carrier of water, 30% propyleneglycol, 5% Ethyl Lactate, 0.1% Azone, and 0.75% hyaluronic acid as agelling agent, with 0.1% neolone 850 as a preservative. An infinitedonor sink model was used wherein 0.75 grams of the drug formulation(1.875 mg of each) was placed in the Franz donor cells. Hanks isotonicsalt solution served as the Franz recipient cell fluid.

HPLC Method: HPLC analysis was performed according to a method developedin our lab. Briefly, a Beckman Coulter system consisting of 125 pumps,508 auto sampler and 168 diode array detector was used with an XTerraRP-C18, 150.times.40 mm, 3 μm column. The analytes were eluted with agradient mobile phase from 25-30% Methanol in HPLC grade water with 0.1%Trifluoroacetic Acid (TFA) and flow rate of 0.7 ml/min. 32 Karat™software (Beckman Coulter, Inc.) was used for acquisition ofchromatograms.

Recipient Cell Concentration Method: The Franz recipient cell balancedsalt solution was analyzed by the HPLC method for MTX concentration.

Histology: Sclera samples were taken and placed in formalin as follows:Normal control sclera with no exposure to drug or light, Franz cell 60minute control drug only exposure and Franz cell 60 minute exposure toboth drug and 350 nm LED light. Fixed slides were evaluated forstructural integrity and gross appearance by a pathologist blinded as tothe origin of the sample tissue.

Statistics: Statistical analysis was performed using one way analysis ofvariance (ANOVA) with Bonferroni's correction. Significance was acceptedat p<0.05.

Results

Recovered MTX concentration from Franz cells experimental results areexpressed as drug quantity per square centimeter (cm²) of exposed tissueover time, e.g., micrograms of MTX recovered from the Franz recipientcell per square centimeter of sclera at each time point (μg/cm²/Time).The human eye volume is only about 6.5 mL, so the ratio of drugpermeation surface available to the internal volume is very largecompared to transdermal drug delivery applications. This surface tovolume ratio indicates that photokinetic in vitro drug flux volumesattained can greatly exceed the required therapeutic range of the drugstested, even with greater than 60 minutes of photokinetic exposure time.

Effect of Light Wavelength on Permeation: For both wavelengths of lighttested all PODD cells showed an improvement of MTX permeation throughthe scleral tissue. In the 350 nm group, significant (p<0.05) levels ofMTX were recovered at the 30 and 60 minute time points (FIG. 8). In the370 nm group, significant (p<0.05) levels of MTX were recovered at the15, 30 and 60 minute time points (FIG. 9). The 370 nm group showed amuch higher flux rate at all time points when compared to the 350 nmgroup.

Histological Examination: Histological examination of the 60 minute PODDexposure compared to passive control revealed no differences between thesamples. The fibrous layers in all samples were grossly intact. No zonesof necrosis were found in the sclera sample areas immediately adjacentto the portion compressed by the Franz cell flange. The two Franz cellmounted sclera samples (60 minute passive control and the PODD)demonstrated no differences from the normal sclera not exposed to drugor light. The structural integrity of the sclera was observed to begrossly intact and undamaged.

DISCUSSION

The data clearly shows that pulsed light of a specific wavelength mayfacilitate methotrexate (MTX) to permeate through ovine sclera atsignificantly higher flux rates when compared to passive controls. Thesharp flux rate differences between the relatively narrow wavelengthsselected (350 nm and 370 nm) for this study further suggests the systemis highly wavelength dependent even within a narrow range.

The energy of light increases as the light wavelength decreases in size;350 nm light has more energy than 370 nm light. The results demonstratethat light with a lower energy caused significantly higher permeationrates. Thus, high light energy in itself is not the deciding factor ofpermeation rates. The absence of sclera damage under histologicalexamination from both light groups along with the differences in fluxrates between within each of the test groups suggests that light energydid not cause unseen physical damage to the fibrous scleral layers thatcould result in permeation pathways. One would suspect that if there wasa physical disruption of the sclera by the light itself, the higherenergy 350 nm light would have higher flux rates. Additionally,incoherent near visible light at 350-370 nm as emitted from an LED hasnot been shown to cause physical damage or disruption to other tissues.

All electronic components generate heat when a current is applied. LEDsdriven by excessive current will get hot and quickly burn out. Whendriven at or slightly below the specified current, exogenous heat isminimal. In early experiments, temperature increases within the Franzdonor cell could not be detected, although we suspect a minor amount ofheat is generated. Again, if heat from the LED was the mechanism ofincreased permeation, then the flux rate of both drugs should have beenincreased in all the wavelength groups.

The use of LEDs as the light source is of convenience. Light emittingdiodes (LED) have inherent narrow wavelength emissions based on thecomposition of the diode material and are available in discretewavelengths of emission and require no further optical filtering. LEDscan be rapidly cycled and switched on and off with no warm up or cooldown light emission phase as in an incandescent bulb. They are veryefficient in converting electrical energy to light energy and producevery little exogenous heat. The actual light emitting portion of a LEDis quite small. The majority of the packaged LED is the housing/lens andelectrical connections. Therefore, the small size, inherent narrow lightwavelength of emission, and efficiency of light production per energyconsumed makes the LED an ideal choice for this system especially forthe limited area of the eye available. Other light sources could be usedif properly optically filtered and controlled for rapid cycle operation.

Light pulse rate may affect the flux rate in the photokinetic system.Unpublished data from early work suggests that high pulse rates (inexcess of 120 cps) actually diminish flux rates. The selection of the24-100 cycles per second pulse rate is based on the inventor's priordata of extensive photokinetic transdermal Franz cell testing of variouslow molecular weight drugs, such as described in U.S. Pat. No. 7,458,982B2.

The selection of Hanks balanced salt solution in the Franz recipientcell rather than HPLC grade water was necessary due to the ability ofthe sclera membrane to regulate osmolarity across this membrane.

Although the total MTX flux did not reach a therapeutic level of 400 μgin this first attempt, various strategies can be employed to increaseflux rates across the sclera barrier membrane such as: optimization ofdrug carrier/chemical permeation enhancement, increasing the drugconcentration in the topical formulation, increasing the exposure timeof the drug on the membrane and increasing the exposed transport area.

Conclusion

The in vitro model demonstrates that pulsed incoherent light of aselected wavelength directed onto a solution of methotrexate applied toovine sclera can be used to facilitate transscleral permeation. Thetransscleral flux rate in the PODD system appears to be wavelengthdependent as determined by spectrophotometry absorption of a subjectdrug. The PODD system did not damage or alter the sclera exposed tolight energy at the wavelengths and intensity used herein. The PODDsystem may be used as an alternative for needle injection into the eye.

Example 2 Delivery of Insulin

Two pertinent ocular drugs were selected for testing the hypotheses.Methotrexate (MW=454 Daltons) is used for the treatment of primaryocular lymphoma. Insulin (MW=5808 Daltons, 5.808 kDa) and insulin likegrowth factors have been implicated as a possible preventive treatmentfor diabetic retinopathy. The transscleral delivery of methotrexate hasbeen described in Example 1, above.

A traditional “up and down” vertical Franz cell (11.28 mm diameterpermeation area, 1.0 cm²) perfusion apparatus (FIG. 6) was adapted andused for this sclera tissue permeation model. PODD modified Franz celltesting device was configured so that it accommodated the placement ofthe selected discreet wavelength LEDs within the donor chamber. DiscreteLEDs at 351 nm and 370 nm were used for the MTX study and 405 nm and 450nm for the insulin study (peak emitting wavelengths at ±10 nm at the 50%radiance output). The LEDs were driven by a square wave pulse generatorset at 24 cycles per second (CPS) with a 50% pulse duration.

Ovine eyes were procured by enucleation within one hour after euthanasiaand placed in tissue culture medium with antibiotics and antimycoticsand refrigerated at 4° C. Sclera tissue was dissected from the eyes andplaced between the donor and recipient chambers of the Franz apparatuswithin 30 hours of enucleation.

Insulin at a concentration of 200 IUs/mL was dissolved in a drug carriercomprised of water, 30% propylene glycol, 5% ethyl lactate, 0.1% Azone,0.75% hyaluronic acid as a gelling agent with 0.1% neolone 850 as apreservative. 0.75 grams of the drug formulation was placed in the Franzdonor cells. Hanks isotonic salt solution served as the Franz recipientcell fluid.

Samples of the Franz recipient solution were taken at 15, 30 and 60minutes of photokinetic exposure and passive controls and analyzed byHPLC for MTX concentration and expressed as μg/cm²/Time. Samples forinsulin were taken at 24 hours and tested by ELISA methodology andexpressed as microunits insulin/cm²/24 hours. The experimentalarrangement is shown in FIG. 6.

Normal control sclera with no exposure to drug or light, Franz cell 60minute control drug only exposure and Franz cell 60 minute exposure toboth drug and 350 nm LED light were taken and placed in formalin. Fixedslides were evaluated for structural integrity and gross appearance by apathologist masked as to the origin of the sample tissue.

Results

For both wavelengths of light tested in the insulin experiments, allPODD cells showed an improvement of insulin permeation through thescleral tissue vs. controls. In both light groups, significant (p<0.05)levels of insulin was recovered at the 24 hour time point (FIG. 10). The450 nm PODD group showed 7 times higher flux rate while the 405 nm groupshowed a 2 times higher flux rate vs. controls. Histological examinationof the 24 hour 405 nm PODD exposure compared to the passive control andnormal sclera not exposed to drug or light revealed no differencesbetween the samples. As was the case in the experiment withmethotrexate, the structural integrity of the sclera was observed to begrossly intact and undamaged.

Discussion

The data clearly shows that pulsed light of a specific wavelength mayfacilitate insulin to permeate through ovine sclera at significantlyhigher flux rates when compared to passive controls. The sharp flux ratedifferences between the relatively narrow wavelengths selected (405 and450 nm for insulin) for this study further suggests the system is highlywavelength dependent even within a narrow wavelength range.

The results demonstrate that light with a lower energy (370 nm vs. 350nm and 450 nm vs. 405 nm) caused significantly higher permeation rates.Thus, high light energy in itself is not the deciding factor ofpermeation rates. The absence of sclera damage under histologicalexamination from the insulin 405 nm light group along with thedifferences in flux rates between within each of the test groupssuggests that light energy did not cause unseen physical damage to thefibrous scleral layers that could result in permeation pathways.Additionally, incoherent near visible light in the 405-450 nm visiblerange as emitted from an LED has not been shown to cause physical damageor disruption to other tissues at the emitting intensities and exposuretimes used herein.

The use of LEDs as the light source is of convenience. Light emittingdiodes (LED) have inherent narrow wavelength emissions based on thecomposition of the diode material and are available in discretewavelengths of emission and require no further optical filtering. LEDscan be rapidly cycled and switched on and off with no warm up or cooldown light emission phase as in an incandescent bulb. They are veryefficient in converting electrical energy to light energy and producevery little exogenous heat. The actual light emitting portion of a LEDis about 300 microns square the remainder is packaging. Therefore, thesmall size, inherent narrow light wavelength of emission, and efficiencyof light production per energy consumed makes the LED an ideal choicefor this system especially for the available limited application area ofthe eye.

Various strategies can be employed to increase total flux rates acrossthe sclera barrier membrane such as: optimization of drugcarrier/chemical permeation enhancement, increasing the drugconcentration in the topical formulation, increasing the exposure timeof the drug on the membrane and increasing the exposed transport area.The available accessible sclera of a human eye is about 4-6 cm². Insulinand insulin like growth factor therapeutic dose requirements are likelyto be very small but with frequent administrations.

Recent photokinetic transdermal permeation studies by the Applicantshave demonstrated significant flux rates of high molecular weighthyaluronic acid (4500 kDaltons) through intact human skin under similarphotokinetic conditions. Scleral tissue is more permeable than humanskin. The practical upper molecular weight limit with the PODD system isunknown and may be determined by the specific molecular configurationrather than molecular weight per se.

Conclusion

The in vitro model demonstrates that pulsed incoherent light of aselected wavelength directed onto a solution of methotrexate or insulinapplied to ovine sclera can be used to facilitate transscleralpermeation without damaging the scleral tissue or chemically alteringthe drug.

Example 3 Transcleral Insulin Delivery of High Molecular Weight Drugs

Using the same procedures as set out above, and the apparatus of FIG. 6,scleral tissue permeation determinations of methotrexate over a shorttime period, as well as the scleral tissue permeation of vancomycin (1mg/mL), insulin, Insulin Like Growth Factor-1 (IGF-1), Avastin™(bevacizumab; Genentech, San Francisco, Calif.), and hyaluronic acid(HA) were conducted. High molecular weight hyaluronic acid (4500 kDa insize) was selected as a test compound because the sodium salt ofhyaluronic acid (SH) is a high molecular weight biopolymer made ofrepeating disaccharide units of glucuronic acid andN-acetyl-β-glucosamine, and which is present in the vitreous body andthe aqueous humor. Hyaluronic acid is a natural polymer which, due toits water retaining capability, binds to cell membranes and cantherefore be considered to be a putative vehicle for controlled oculardelivery (Durrani, et al., Int. J. Pharm., Vol. 118 (2), p. 243-250(1995)). The results are shown in FIGS. 11-16, and are summarized inFIG. 17. As can be seen from FIG. 17, significant transscleral flux isobserved with molecules ranging from about 450 Daltons (methotrexate) tomolecules with molecular weights of about 4500 K Da (hyaluronic acid,HA). In addition to the significant and therapeutic transcleral fluxesillustrated by the methods and apparatus of the present disclosure, thepresent photokinetic system significantly increases the intrascleraldeposition of compounds as well. This in turn allows for the scleraitself to become a depot for extended drug release into the intravitrealspace, as well as the eye circulation.

Example 4 Transcleral Insulin Delivery Rabbit Model

A proof-of-concept experiment was conducted for the PODD system of thepresent disclosure with a rabbit ocular drug delivery model using afluorescent labeled human insulin molecule as the test drug for a60-minute photokinetic exposure. Exogenous human insulin can bedifferentiated from the endogenous rabbit insulin; the human insulinFITC (fluorescein isothiocyanate) fluorescent tag further allows fortracking of the drug within the tissues. FITC-labeled human insulin(5733 Daltons in size, available from Invitrogen Corporation, Carlsbad,Calif. as insulin modified at the N-terminus of the B-chain with an FITCconjugate/tag) was used as a test drug for transocular applications, asthe ELISA analytical methods are widely available and accepted toprovide a quantitative as well as a functional assay for the molecule.Franz permeation cells as described herein were utilized to definefunctional photokinetic parameters of light wavelength and pulse rate invitro prior to the animal study.

In this series, rabbits were fitted with transscleral photokineticdevice, such as shown in FIG. 1 of this disclosure, for one hour. Oneeye of the test rabbit was exposed to the topically applied insulin, 4micro-units/mL in a suitable drug carrier formulation under an adaptedphotokinetic device using 450 nm LED light pulsed at 100 CPS for onehour, applied to a 2 cm² area of sclera. Images were taken of theposterior optic disc, the area where the retina, optic nerve and bloodvessels come together on the posterior segment of the eye. These resultsare shown in FIGS. 18A, 18B, and 18C, with FIG. 18A illustrating thefluorescence baseline image, FIG. 18B illustrating an IR baseline image,and FIG. 18C illustrating the fluorescence image after 1 hour of PODD.These rabbit optic disc images demonstrate that the fluorescent taggedhuman insulin administered with the PODD system of the present systemreached the posterior segment of the rabbit's eye. The contralateral eyeshowed no fluorescence. Passive permeation in other rabbits with thesame experimental setup did not show fluorescence in the optic disc.

After treatment for one hour with the transscleral photokinetic devicedescribed herein, and after the images in FIGS. 18A-18C were taken, theanimals were sacrificed and tissues and fluids were taken forquantitative analysis. The tissues from the photokinetic exposed eye andthe contralateral eye that were assayed for human insulin concentration.A passive permeation control animal without light exposure but underotherwise similar conditions was also assayed. The results of threeconcentration of human FITC labeled insulin in the PODD device vs.passive permeation of 4 mU/mL in the various fluids and tissues of therabbit eye after 60 minute exposure are presented in FIGS. 19A-19F, andin Table 1, below. Although the ELISA analytical test method employed isspecific for human insulin, there is some cross reactivity with rabbitinsulin (“No Ins” in the Figures) as the kit contains rabbit proteins aspreservatives. A small quantity of human insulin was also found in theuntreated contralateral eye transferred by the blood circulation, asalso evidenced in the figures.

TABLE 1 Insulin (μU/mL) Insulin (μU/gram Tissue/mg Protein) VitreousAqueous RPE Neural Humor Humor Sclera Cornea Choroid Retina Photokineticexposure 118 114 1498 1500 31 28 Photokinetic Contralateral 6 40 98 223.75 2.5 Passive Delivery 4 2 200 200 0.89 1.3 Passive Contralateral 2 256 22 0.65 0.98

In view of the results shown in Table 1, this test demonstrated thefeasibility of the photokinetic transcleral and/or transcorneal drugdelivery system as described herein. The photokinetic transcleraldelivery provided drug concentrations within the several ocularcompartments.

Other and further embodiments utilizing one or more aspects of theinventions described above can be devised without departing from thespirit of Applicant's invention. Further, the various methods andembodiments of the disclosure can be included in combination with eachother to produce variations of the disclosed methods and embodiments.Discussion of singular elements can include plural elements andvice-versa.

The order of steps can occur in a variety of sequences unless otherwisespecifically limited. The various steps described herein can be combinedwith other steps, interlineated with the stated steps, and/or split intomultiple steps. Similarly, elements have been described functionally andcan be embodied as separate components or can be combined intocomponents having multiple functions.

The inventions have been described in the context of preferred and otherembodiments and not every embodiment of the invention has beendescribed. Obvious modifications and alterations to the describedembodiments are available to those of ordinary skill in the art. Thedisclosed and undisclosed embodiments are not intended to limit orrestrict the scope or applicability of the invention conceived of by theApplicants, but rather, in conformity with the patent laws, Applicantsintend to fully protect all such modifications and improvements thatcome within the scope or range of equivalent of the following claims.

1. A method for the photokinetic transscleral ocular delivery of abiologically active substance to a subject, the method comprising:preparing a solution comprising the biologically active substance and asolvent; applying the solution to an exterior ocular surface of an eyeof the subject; and illuminating the solution on the exterior ocularsurface with a pulsed incoherent light directed through the solution andtowards an interior of the eye of the subject at a wavelength selectedto induce photokinetic transocular translocation of the biologicallyactive substance.
 2. The method of claim 1, wherein the solution furthercomprises a gelling agent.
 3. The method of claim 1, wherein thebiologically active substance has a molecular weight greater than 500Daltons.
 4. The method of claim 1, wherein the biologically activesubstance is selected from one or more anesthetics, anti-infectives,antibiotics, antifungals, antivirals, antineoplastics, anti-VEGFs,antineovasculars, steroids, anti-inflammatories, immunomodulators,gases, antioxidants, nanoparticles, genes, cytokines, peptides,antithrombotics, nucleotides, RNAs, anti-compliment medications,compliment modulating medications, peptides, immunoglobulins,antibodies, antigens, anti-glaucoma medications, hormones, vitamins,amino acids, silicone liquids, heavy liquid tamponades, cellularnutrients, anti-apoptotic agents, anticoagulants, tissue adhesives,cofactors, coenzymes, enzymes and combinations thereof.
 5. The method ofclaim 4, wherein the hormone is selected from the group consisting ofmethionine enkephalin acetate, leucine enkephalin, angiotensin IIacetate, β-estradiol, methyl testosterone, methyl prednisolone,corticosteroids, budenoside, progesterone, insulin, and combinationsthereof.
 6. The method of claim 4, wherein the biologically activesubstance is insulin.
 7. The method of claim 4, wherein the biologicallyactive substance is triamcinolone acetonide.
 8. The method of claim 4,wherein the antibody is a monoclonal antibody selected from the groupconsisting of adalimumab, bevacizumab, daclizumab, etanercept,infliximab, ranibizumab, rituximab, and combinations thereof.
 9. Themethod of claim 4, wherein the antibiotic is selected from the groupconsisting of amoxycillin, ampicillin, penicillin, clavulanic acid,aztreonam, imipenem, streptomycin, gentamicin, vancomycin, clindamycin,ephalothin, erythromycin, polymyxin, bacitracin, amphotericin, nystatin,rifampicin, tetracycline, coxycycline, chloramphenicol, zithromycin, andcombinations thereof, as well as pharmaceutically acceptable derivativesthereof.
 10. The method of claim 1, wherein the biologically activesubstance is insulin-like growth factor-1 (IGF-1).
 11. The method ofclaim 4, wherein the antiviral agent is selected from the groupconsisting of guanosine derivatives, nucleoside phosphonates, phosphonicacid derivatives, oligonucleotides, and combinations thereof, as well aspharmaceutically acceptable salts, solvates, hydrates, and derivativesthereof.
 12. The method of claim 4, wherein antiviral drug is selectedfrom the group consisting of foscarnet, fomivirsen sodium, trifluridine,vidarabine, and combinations thereof, as well as pharmaceuticallyacceptable derivatives thereof.
 13. The method of claim 1, wherein thebiologically active substance is methotrexate.
 14. The method of claim2, wherein the gelling agent selected from the group consisting ofhydroxyethyl cellulose, pectines, agar, alginic acid and its salts, guargum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose, propylenecarbonate, polyethylene glycol, hexylene glycol sodiumcarboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropyleneblock copolymers, pluronics, wood wax alcohols, tyloxapol, andcombinations thereof, as well as pharmaceutically acceptable derivativesthereof.
 15. The method according to claim 1, wherein the solvent is anaqueous solvent or an organic solvent.
 16. The method according to claim15, wherein the aqueous solvent is an aqueous solution of ethyl lactateor propylene glycol.
 17. The method according to claim 1, wherein thepulsed incoherent light is selected from the group consisting offluorescent, ultraviolet, visible, near infrared, LED (light emittingdiode), and halogen light.
 18. The method according to claim 1, whereinthe wavelength is in a range from about 260 nm to about 760 nm.
 19. Themethod according to claim 1, wherein the wavelength is in a range fromabout 340 nm to about 900 mm.
 20. The method according to claim 1,wherein the wavelength is selected from the group consisting of 350 nm,370 nm, 390 nm, 405 nm, 450 nm and combinations thereof.
 21. The methodaccording to claim 1, wherein the pulsed incoherent light is applied ata pulse rate between about 1.7 cycles per second (cps) and about 120cps.
 22. The method according to claim 21, wherein the pulse rate isbetween about 1.7 cps and about 80 cps.
 23. The method according toclaim 1, wherein the pulsed incoherent light is applied at a duty cycleis between about 50% and about 75%.
 24. A method for the transcleraldelivery of one or more high molecular weight biologically activesubstances to the eye of a subject, the method comprising: preparing asolution comprising a biologically active substance having a molecularweight greater than 1000 Daltons and a solvent; applying the solution toan exterior ocular surface of a subject; illuminating the solution onthe exterior ocular surface with a pulsed incoherent light directedthrough the solution and towards an interior of the eye of the subjectat a wavelength selected to induce photokinetic transoculartranslocation of the biologically active substance.
 25. The method ofclaim 24, wherein the biologically active substance has a molecularweight of greater than 50,000 Daltons.
 26. The method of claim 24,wherein the biologically active substance is insulin, bevacizumab, humangrowth factor, and combinations and/or pharmaceutically acceptablesalts, solvates, or derivatives thereof.
 27. The method of claim 24,wherein the biologically active substance is selected from the groupconsisting of anti-infectives, antibiotics, antifungals, antivirals,antineoplastics, anti-VEGFs, antineovasculars, steroids,anti-inflammatories (including NSAIDS), immunomodulators, antioxidants,nanoparticles, genes, cytokines, peptides, antithrombotics,polynucleotides, RNAs, anti-compliment medications, complimentmodulating medications, immunoglobulins, antibodies, antigens,anti-glaucoma medications, hormones, vitamins, silicone liquids, heavyliquid tamponades, cellular nutrients, anti-apoptotic agents,anticoagulants, tissue adhesives, cofactors, coenzymes, enzymes andcombinations thereof.
 28. A method for the treatment of a subject havinga VEGF-related angiogenic disease affecting the eyes of the subject, themethod comprising administering to a subject in need thereof atherapeutically effective amount of a biologically active substanceusing the method of claim 1, wherein the VEGF-related angiogenic diseaseis selected from the group consisting of cancer, age-related maculardegeneration (AMD), diabetic retinopathy, and combinations thereof.